The present invention relates to a new method to rapidly identify genes that function in non-host resistance. It also relates to genes identified by this method that enhance levels of disease resistance if expressed in susceptible plants.
Genetic diversity is an important factor in the balanced evolution between plants and their pathogens. In natural systems, outbreeding plant populations interact with mixed pathogen populations. This interaction is often dependent on the presence of resistance (R-) genes in the plant and avirulence (avr) genes in the pathogen. The outbreeding plants share pools of R-genes, and the plant pathogens produce a variety of elicitors, directly or indirectly produced by the avr genes. Individual plants that contain R-genes that somehow recognize one of the elicitors produced by an infecting pathogen are resistant against this pathogen.
R-gene mediated resistance usually results in a hypersensitive response (HR), observed as rapid necrosis at the infection site. Apparently, the activated R-gene triggers a signal transduction event leading to apoptotic cell death, which may prevent the invading pathogen from spreading beyond the infection site and trigger resistance in non-infected adjacent cells.
Over the last five years, a number of R-genes have been cloned. The most ubiquitous class of R-genes encode proteins with a C-terminal leucine rich repeat, an N-terminal nucleotide binding site, and a conserved stretch of amino acids with the consensus sequence GLPLAL. Examples of this class of R-genes are Rps2 (Bent et al., 1994), N (Whitham et al., 1994), L6 (Lawrence et al., 1995), M (Anderson et al., 1997), and Rpm1 (Grant et al., 1995). Progress has also been made in the identification of proteins involved in R-gene mediated signal transduction. Recent papers report the involvement of protein kinases, putative transcription factors, and lipase-like proteins in R-gene signalling (reviewed by Innes, 1998). Recently, it has been shown that the engineering of these signaling components may also lead to enhanced levels of disease control in plants (Cao et al., 1998).
It is believed that R-genes do not provide protection against all genotypes of a pathogen, i.e., pathogens within a species do not all produce the same elicitor. It is therefore likely that infections of outbreeding populations will result in the survival of part of the population only. Modem agriculture may likely disturb the balance between plants and pathogens. Outbreaks of a disease that several decades ago would impact a relatively limited number of plants can now cause devastating epidemics.
To prevent major losses to diseases, plant breeders attempt to introgress resistance against the most important pathogen races into elite cultivars. In most cases, this is a never-ending battle because resistance against one or several genotypes of a pathogen will select for occurrence of other genotypes. For example, the subsequent introgression of eleven R-genes from the resistant wild potato species Solanum demissum into cultivated susceptible potato cultivars resulted in all cases in the emergence of virulent genotypes of the pathogen Phytophthora infestans. Classical breeding is by definition based on crossing programs and, therefore, can only transfer resistance traits between different accessions or cultivars of the same plant species or between plant species that are sexually compatible. This resistance is often referred to as xe2x80x9chostxe2x80x9d resistance. Temporal control of many pathogens including the following have been obtained by introgression of R-genes: Phytophthora infestans, Phytophthora megasperna, Puccinia graminis, Puccinia recondita, Puccinia sorghi, Puccinia coronata, Puccinia helianthi, Puccinia striiformis, Erysiphe graminis, Ustilago hordei, Ustilago avenae, Uromyces phaseoli, Peronospora farinosa, Pseudomonas syringae, Xanthomonas oryzae, Cladosporium fulvum, brown plant hopper, aphids, hessian fly, and tobacco mosaic virus.
A few R-genes have been identified that provide resistance against most races of a particular pathogen. Of particular interest are the rice Xa21 gene that controls most races of Xanthomoas oryzae (Mazzola et al., 1994; Song et al., 1995), the wheat Lr34 gene involved in resistance to most leaf rusts, and the barley Rpg1 gene that protects plants against almost all stem rusts. However, these R-genes are rare and may be broken by new aggressive races.
A superior source of resistance that provides broad-spectrum and durable disease control but is unaccessible to classical breeding is the so-called xe2x80x9cnon-hostxe2x80x9d resistance. A plant species displays non-host resistance if all sexually compatible accessions and cultivars of that particular species or very related species are resistant to all genotypes of a particular pathogen. Due to the lack of susceptible material within those plant species, it is impossible to determine the genetic basis of non-host resistance.
To date, no genes have been cloned that are known to be involved in active non-host resistance. However, it is possible that such genes resemble the R-genes isolated from sources displaying host resistance. Support for this hypothesis comes from studies on the interaction between P. infestans and the non-host plant species Nicotiana tabacum (tobacco). The resistance of tobacco correlates with its ability to respond with an HR to infection, suggesting that the resistance of tobacco against P. infestans is based on an active defense mechanism controlled by R-genes (Kamoun et al., 1997). Thus, the non-host resistance of tobacco appears to be xe2x80x9cactivexe2x80x9d, and is different from xe2x80x9cpassivexe2x80x9d resistance that is based on factors such as the presence of preformed pathogen inhibitors or the absence of factors that are essential for pathogen growth (Ride, 1985).
It can be envisioned that expression of certain cloned non-host resistance genes in susceptible crops would provide the broad-spectrum and durable disease resistance levels that are needed in modern agriculture. However, it is impossible to isolate non-host resistance genes through genetics-based methods. Here, the inventors have developed a new technique based on the isolation and screening of large numbers of genes that are associated with active non-host resistance. The screening is performed in plants that are both susceptible to certain target pathogens and highly accessible to transformation. By implementing this technique, a number of genes have been identified that enhance, or are expected to enhance, levels of disease resistance if expressed in susceptible plants.
The present invention relates to a method to screen genes associated with non-host resistance for those genes that enhance levels of resistance if expressed in susceptible plants, by transforming tissue of a pathogen-susceptible plant with these genes, challenging the transformed tissue with a pathogen or its elicitor, and observing enhanced defense and/or HR responses. In a particular embodiment of the invention, homologs of R-genes from tobacco are identified by gene amplification, cotransformed with the INF1 elicitor of Phytophthora infestans into leaves of Nicotiana benthamiana, and screened for the presence of a hypersensitive response, which indicates functionality. In another embodiment, genes associated with non-host resistance are identified by first selecting genes that are induced by target pathogens in the non-host but not (or not as much) in susceptible hosts, and second screening them for their ability to enhance resistance against a model pathogen such as the bacterial pathogen Pseudomonas tabaci if transiently overexpressed in leaves of N. benthamiana plants.
In one aspect, the present invention provides novel nucleic acid sequences (SEQ ID NO:57 and SEQ ID NO: 1-10 and SEQ ID NO:58, 60, 62) that can confer disease resistance to Phytophthora infestans to plants. A further embodiment of the invention provides novel protein sequences (SEQ ID NO: 11-20 and SEQ ID NO:59) involved in disease resistance to Phytophthora infestans in plants.
In a further embodiment of the invention, plant cells or transgenic plants comprising a nucleic acid sequence conferring enhanced resistance to Phytophthora infestans are provided as well as seed or progeny from such plants. A transgenic plant, seed, or progeny thereof that comprises a nucleic acid sequence of SEQ ID NO:57 displays resistance to disease from or a hypersensitive response in response to Phytophthora infestans or other fungal pathogens as compared to an otherwise similar plant lacking the nucleic acid sequence. A transgenic plant, seed, or progeny thereof that comprises a nucleic acid sequence of SEQ ID NO:60 displays resistance to disease from or a hypersensitive response in response to Phytophthora infestans or other fungal pathogens as compared to an otherwise similar plant lacking the nucleic acid sequence. Also provided are related methods of producing a transgenic plant exhibiting enhanced resistance to fungal pathogens comprising introducing into a plant cell a nucleic acid sequence encoding an R-protein thereby producing a transformed cell, and regenerating a transgenic plant therefrom that displays resistance to a selected fungal pathogen or pathogens as compared to an otherwise similar plant lacking the nucleic acid sequence.
The present invention also encompasses the use of any of the DNA sequences or biologically functional equivalents thereof disclosed herein to produce recombinant plasmids, transformed microorganisms, probes, molecular markers, and primers useful to identify related nucleic acid sequences that confer resistance to fungal pathogens on plant cells and to produce transgenic plants resistant to such fungal pathogens.
The foregoing and other aspects of the invention will become more apparent from the following detailed description and accompanying drawings.